Suppression of myeloid derived suppressor cells and immune checkpoint blockade

ABSTRACT

Impressive responses have been observed in patients treated with checkpoint inhibitory anti-PD-1 or anti-CTLA-4 antibodies. However, immunotherapy against poorly immunogenic cancers remains a challenge. Treatment with both anti-PD-1 and anti-CTLA-4 antibodies were unable to eradicate large, modestly immunogenic CT26 tumors or metastatic 4T1 tumors. However, co-treatment with epigenetic modulating drugs and checkpoint inhibitors markedly improved treatment outcomes, curing more than 80% of them. Functional studies revealed that the primary targets of the epigenetic modulators were myeloid-derived suppressor cells (MDSCs). A PI3K-inhibitor that reduced circulating MDSCs also cured 80% of mice with metastatic 4T1 tumors when combined with immune checkpoint inhibitors. Thus, cancers resistant to immune checkpoint blockade can be cured by eliminating MDSCs.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grants CA043460and CA062924 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

TECHNICAL FIELD OF THE INVENTION

This invention is related to the area of cancer treatment. Inparticular, it relates to combination therapies to overcome treatmentrefractory tumors.

BACKGROUND OF THE INVENTION

The mammalian immune system is delicately regulated, allowing it tomount an effective attack against foreign invaders such as bacteria andviruses with minimal bystander casualties. This requires functionallyredundant regulatory mechanisms to ensure safety (1-3). Cancers appearable to hijack these mechanisms to avoid immune destruction. Several ofthe regulatory mechanisms exploited by cancer have been identified.These include regulatory T cells (Tregs), circulating MDSCs, residenttumor-associated macrophages and neutrophils, checkpoint inhibitingreceptors on T-cells, and immunosuppressive cytokines (4-8). Mostrecently, the checkpoints guarded by the PD-1 and CTLA-4 receptors havebeen under intense investigation because of the availability ofantibodies which can inhibit their function. Recent clinical trials withanti-CTLA-4, anti-PD-1 and anti-PD-L1 monoclonal antibodies (mAbs)showed remarkable therapeutic responses (9-12), underscoring the ideathat disruption of the immune checkpoints can be therapeutically useful.Yet, the objective responses were observed in a minority of the treatedpatients and tumor types, and the reasons why certain tumors respond andothers don't are mysterious. CT26 and 4T1 are among the most popularsyngeneic tumor models used for assessing novel therapeutic approaches.CT26 was derived from an undifferentiated colorectal carcinoma inducedin a BALB/c mouse by repeated intrarectal instillations ofN-nitroso-N-methylurethan and shown to be modestly immunogenic (13, 14),whereas 4T1 originated from a spontaneous mammary tumor in a BALB/cmouse (15). 4T1 is poorly immunogenic and highly metastatic,characteristics shared with advanced human cancers (16). Despite theextensive use of these tumor cell lines in cancer research, littlegenetic characterization is available for either of them.

There is a continuing need in the art to overcome the problem oftreatment recalcitrant cancers so that remissions may be longer lastingand more widely spread in the population treated.

SUMMARY OF THE INVENTION

According to one aspect of the invention a method of treating atumor-bearing mammal is provided. At least one first agent whichsuppresses myeloid derived suppressor cells (MDSCs) is administered tothe mammal. At least one second agent which blocks an immune checkpointis administered to the mammal. The tumor may or may not be non-smallcell lung cancer (NSLC).

According to another aspect of the invention a kit is provided. The kitcomprises a single package and contains at least one first agent whichsuppresses myeloid derived suppressor cells (MDSCs). It further containsat least two second agents which block at least two immune checkpoints.

In yet another aspect of the invention a composition is provided. Thecomposition comprises at least one first agent which suppresses myeloidderived suppressor cells (MDSCs) and at least two second agents whichblock at least two immune checkpoints.

According to another aspect of the invention a method of treating atumor-bearing mammal is provided. At least one first agent whichsuppresses myeloid derived suppressor cells (MDSCs) is administered tothe mammal. At least two second agents which block at least two immunecheckpoints are administered to the mammal.

In yet another aspect of the invention a method of treating a mammalwith a bacterial or viral infection is provided. At least one agentwhich suppresses myeloid derived suppressor cells (MDSCs) isadministered. The agent is selected from the group consisting of ahistone deacetylase inhibitor, a DNA methyltransferase inhibitor, ap110α subunit of phosphoinositol 3 kinase (PI3K) inhibitor, andcombinations thereof.

These and other embodiments which will be apparent to those of skill inthe art upon reading the specification provide the art with therapeuticpreparations and methods for treating hard to treat tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1G. Therapeutic response of tumor-bearing mice. BALB/c micebearing different tumors were treated with various therapeuticmodalities as indicated. IgG, IgG control; P, anti-PD-1 antibody; C,anti-CTLA-4 antibody; AZA, 5-azacytidine; ENT, entinostat. Tumor volumes(FIG. 1A, FIG. 1C, and FIG. 1E) and animal survival (FIG. 1B, FIG. 1D,and FIG. 1F) were recorded. (FIG. 1A and FIG. 1B) BALB/c mice with CT26tumors of moderate sizes. (FIG. 1C and FIG. 1D) BALB/c mice with largeCT26 tumors. (FIG. 1E and FIG. 1F) BALB/c mice with metastatic 4T1tumors. (FIG. 1G) 4T1 tumor-bearing mice were treated as indicated andeuthanized six weeks after tumor implantation. Primary tumor from eachmouse was measured and metastatic lesions (mets) lesions in differentorgans were counted. Means and standard deviations are shown. Number (n)of animals used in each experimental arm and P-values are alsoindicated. *P<0.05, **P<0.01, ***P<0.001, ns, not significant.

FIGS. 2A-2H. Response of immune cells following immune checkpointblockade and epigenetic modulation. BALB/c mice bearing metastatic 4T1tumors were treated with indicated therapeutic modalities, followed byFACS and immunohistofluorescent analyses to assess tumor-infiltratingand circulating immune cells. Means and standard deviations are shown,with P-values indicated. (FIG. 2A) FACS result for tumor-infiltratingCD8⁺ T cells. (FIG. 2B) Representative immunohistofluorescent stainingof tumor-infiltrating CD8⁺ T cells. Scale bar, 50 μm. (FIG. 2C) FACSresult for tumor-infiltrating CD4⁺CD25⁺FoxP3⁺ Tregs. (FIG. 2D)Representative FACS data showing percentages of FoxP3 and CD25 doublepositive cells in CD45⁺CD3⁺CD4⁺ gated tumor-infiltrating cells. (FIG.2E) FACS result for circulating G-MDSCs. (F) Representative FACS datashowing percentages of Ly6G⁺Ly6C^(lo) cells in CD45⁺CD11b⁺F4/80⁻MHC-II⁻gated circulating cells. (FIG. 2G) FACS result for tumor-infiltratingG-MDSCs. (FIG. 211) Representative immunohistofluorescent staining oftumor-infiltrating Ly6G⁺ cells. Scale bar, 50 μm.

FIGS. 3A-3C. Myeloid-derived Ly6G⁺ cells are responsible for resistanceto immune checkpoint blockade. (FIG. 3A) BALB/c mice bearing 4T1 tumorswere treated with various antibodies or antibody combinations asindicated and tumor volumes recorded over time. αLy6G, anti-Ly6Gantibody; αCD25, anti-CD25 antibody. (FIG. 3B) FACS result forcirculating G-MDSCs after treatment with different antibodies orantibody combinations. (FIG. 3C) 4T1 tumor-bearing mice were treatedwith anti-PD-1/anti-CTLA-4 antibodies plus epigenetic modulators with orwithout adoptive transfer of MDSCs isolated by affinity purificationfrom the 4T1 tumor-bearing animals. Tumor volumes were recordedfollowing the treatments. Means and standard deviations are shown, withP-values indicated.

FIGS. 4A-4D. Direct effects of epigenetic modulators on cultured cells.(FIG. 4A and FIG. 4B) 4T1 cells, purified CD8⁺ T cells or G-MDSCs weretreated with different concentrations of entinostat (FIG. 4A) or AZA(FIG. 4B). Cell viability was assessed using a metabolism-basedcolorimetric assay. (FIG. 4C) Conditioned media from co-cultures ofG-MDSCs and CD8⁺ T cells at different ratios were analyzed for IFN-γconcentration. (FIG. 4D) Conditioned media from co-cultures at a G-MDSCto CD8⁺ T cell ratio of 1:1 were collected after treatment withentinostat at increasing doses for 24 hours and analyzed for IFN-γconcentration. Means and standard deviations of data from at leasttripli cate wells are shown. P-values are indicated.

FIG. 5 (S1). Body weight measurements. BALB/c mice bearing 4T1 tumorswere treated with indicated therapeutic modalities. Their body weightswere measured and recorded regularly after treatments. Means andstandard deviations of data are shown.

FIG. 6 (S2). Expression of genes involved in MHC-I presentation. RNA wasisolated from 4T1 and CT26 tumor cells cultured in vitro after treatmentwith the epigenetic modulators. The expression of different genesinvolved in MHC-I presentation was assessed by RT-PCR. β-actin was usedas loading control. Lane A, untreated, Lane B, AZA treated, Lane C,entinostat treated, Lane D, AZA/entinostat treated.

FIGS. 7A-7B. (S3). Elevated G-MDSC levels induced by 4T1 tumor. (FIG.7A) Peripheral blood was collected at indicated time points after 4T1tumor implantation and analyzed for the levels of G-MDSCs by FACS. (FIG.7B) Peripheral blood, spleen, and tumor were harvested from healthy miceor from tumor-bearing mice on day 18 after 4T1 tumor implantation andanalyzed for the levels of G-MDSCs by FACS. Means and standarddeviations of data are shown.

FIGS. 8A-8C (S4). PI3K inhibitor eradicates 4T1 tumor by depletingG-MDSC when combined with immune checkpoint blockade. (FIG. 8A) G-MDSCs,CD8⁺ T cells and 4T1 tumor cells were treated in vitro with J32 atvarious concentrations. Cell viability was assessed using ametabolism-based colorimetric assay. Means and standard deviations ofdata from triplicate wells are shown. (FIG. 8B) BALB/c mice bearing 4T1tumors were treated with indicated therapeutic modalities. FACS analysiswas performed to quantify circulating G-MDSCs. (FIG. 8C) BALB/c micebearing 4T1 tumors were treated with J32, anti-PD-1/anti-CTLA-4antibodies or the combination and tumor volumes recorded. Means andstandard deviations are shown, with P-values indicated.

FIG. 9. Meier Kaplan curves for combination treatments. From lowest tohighest curves: C. novyi-NT alone; Anti-PD-1/anti-CTLA-4 alone;Anti-PD-1/anti-CTLA-4+C. novyi-NT; Anti-PD-1/anti-CTLA-4+ENT/AZA;Anti-PD-1/anti-CTLA-4+ENT/AZA+C. novyi-NT.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have developed a therapeutic approach that involves agentsthat act on host cells in the immune system such as MDSCs to reduceand/or suppress them. Such agents may be epigenetic modulators such ashistone deacetylase inhibitors or DNA methyltransferase inhibitors. Whenused in conjunction with immune checkpoint blockade, the epigeneticmodulators kill MDSCs at much lower concentrations than required forkilling tumor cells in vitro. The epigenetic modulators have only amarginal effect at best on tumor cells in vivo at the doses used;reduction of MDSCs using antibodies directed against them has similarantitumor effects to those observed with the epigenetic modulators. Inadoptive transfer experiments, MDSCs purified from non-treatedtumor-bearing mice can abolish the therapeutic effects of epigeneticmodulation. Inhibition of MDSCs with a completely different class ofagents (a PIK3 inhibitor) has similar effects to those of epigeneticmodulators.

Types of tumors which are amenable to treatment according to the methodsof the invention and/or using the kits and/or using the compositions ofthe invention are both solid tumors and hematological cancers. Exemplarytumors include Adrenal Cancer, Anal Cancer, Bile Duct Cancer, BladderCancer, Bone Cancer, Brain/CNS Tumors In Adults, Brain/CNS Tumors InChildren, Breast Cancer, Breast Cancer In Men, Cancer in Adolescents,Cancer in Children, Cancer in Young Adults, Cancer of Unknown Primary,Castleman Disease, Cervical Cancer, Colon/Rectum Cancer, EndometrialCancer, Esophagus Cancer, Ewing Family Of Tumors, Eye Cancer,Gallbladder Cancer, Gastrointestinal Carcinoid Tumors, GastrointestinalStromal Tumor (GIST), Gestational Trophoblastic Disease, HodgkinDisease, Kaposi Sarcoma, Kidney Cancer, Laryngeal and HypopharyngealCancer, Leukemia, Leukemia—Acute Lymphocytic (ALL) in Adults,Leukemia—Acute Myeloid (AML), Leukemia—Chronic Lymphocytic (CLL),Leukemia—Chronic Myeloid (CML), Leukemia—Chronic Myelomonocytic (CMML),Leukemia in Children, Liver Cancer, Lung Cancer, Lung Cancer—Non-SmallCell, Lung Cancer—Small Cell, Lung Carcinoid Tumor, Lymphoma, Lymphomaof the Skin, Malignant Mesothelioma, Multiple Myeloma, MyelodysplasticSyndrome, Nasal Cavity and Paranasal Sinus Cancer, NasopharyngealCancer, Neuroblastoma, Non-Hodgkin Lymphoma, Non-Hodgkin Lymphoma InChildren, Oral Cavity and Oropharyngeal Cancer, Osteosarcoma, OvarianCancer, Pancreatic Cancer, Penile Cancer, Pituitary Tumors, ProstateCancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer,Sarcoma—Adult Soft Tissue Cancer, Skin Cancer, Skin Cancer—Basal andSquamous Cell, Skin Cancer—Melanoma, Skin Cancer—Merkel Cell, SmallIntestine Cancer, Stomach Cancer, Testicular Cancer, Thymus Cancer,Thyroid Cancer, Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer,Waldenstrom Macroglobulinemia, and Wilms Tumor.

Types of bacterial infections which can be treated according to themethods of the invention include Bacillus anthraces, Bordetellapertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis,Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydiapneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridiumbotulinum, Clostridium difficile, Clostridium perfringens, Clostridiumtetani, Corynebacterium diphtheriae, Enterococcus faecalis andEnterococcus faecium, Escherichia coli (generally), EnterotoxigenicEscherichia coli (ETEC), Enteropathogenic E. coli, E. coli O157:H7,Francisella tularensis, Haemophilus influenzae, Helicobacter pylori,Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes,Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae,Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa,Rickettsia rickettsia, Salmonella typhi, Salmonella typhimurium,Shigella sonnei, and Staphylococcus aureus.

Types of viral infections which can be treated according to theinvention include both chronic and acute infections. Exemplary infectioninclude Respiratory Viruses, such as, Adenoviruses, Avian influenza,Influenza virus type A, Influenza virus type B, Measles, Parainfluenzavirus, Respiratory syncytial virus (RSV), Rhinoviruses, SARS-CoV,Gastro-enteric Viruses, such as, Coxsackie viruses, Enteroviruses,Poliovirus, Rotavirus, Hepatitis Viruses, such as, Hepatitis B virus,Hepatitis C virus, Bovine viral diarrhea virus (surrogate), HerpesViruses, such as, Herpes simplex 1, Herpes simplex 2, Humancytomegalovirus, Varicella zoster virus, Retroviruses, such as, Humanimmunodeficiency virus 1 (HIV-1), Human immunodeficiency virus 2(HIV-2), Simian immunodeficiency virus (SIV), Simian humanimmunodeficiency virus (SHIV), Viral Select Agents/Emerging ViralPathogens, such as, Avian influenza, Dengue virus, Hantavirus,Hemorrhagic fever viruses, Lymphocytic choromeningitis virus, Smallpoxvirus surrogates, Cowpox, Monkeypox, Rabbitpox, Vaccinia virus,Venezuelan equine encephalomyelitis virus (VEE), West Nile virus, Yellowfever virus.

Types of antibodies which may be used to target immune checkpoints orMSDCs may be of any isotype. They may be humanized or chimeric or othermammal or animal. They may be conjugated to other moieties such astoxins. They may be monoclonal or polyclonal. They may be single chainantibodies, fragments, or portions of antibodies.

Modes of administration for the therapeutic agents may be those whichare used in the art. Examples include oral, topical, inhalation, andinjection. Locations for administration include epicutaneous or topical,nasal administration, intraarterial, intraarticular, intracardiac,intramuscular, intradermal, intralesional, intraosseous infusion,intraperitoneal, intrathecal, intrauterine, intravaginal administration,intravenous, Intravesical infusion, intravitreal, subcutaneous,transdermal, transmucosal, avitreal, subcutaneous, transdermal, andtransmucosal.

Mammals which may be treated include any which are susceptible totumors, hematological cancers, bacterial infections or viral infections.These include: Acouchi—Myoprocta acouchy (formerly, pratti), Alpaca—Lamapacos, Anteater, giant—Myrmecophaga tridactyla, Armadillo, southern(formerly La Plata) three-banded Tolypeutes matacus, Bandedmongoose—Mungos mungo, Bear, sloth—Melursus ursinus, Bear, Andean orspectacled—Tremarctos ornatus, Beaver, American—Castor canadensis,Bison, American—Bison bison (SCBI Front Royal only), Caracal—Caracalcaracal, Cat, fishing—Prionailurus viverrinus, Cavy, rock—Kerodonrupestris, Cheetah—Acinonyx jubatus, Clouded leopard—Neofelis nebulosa,Coati—Nasua narica, Colobus, black-and-white or guereza—Colobus guereza,Cow, Hereford—Bos taurus taurus hereford, Cow, Holstein—Bos taurustaurus holstein, Deer, Burmese brow-antlered (Eld's deer)—Cervus eldithamin (SCBI Front Royal only), Deer, tufted—Elaphodus cephalophus (SCBIFront Royal only), Degu—Octodon degus, Donkey, miniature—Equus asinusasinus miniature, Elephant, Asian—Elephas maximus, Elephant shrew,short-eared—Macroscelides proboscideus, Ferret, black-footed—Mustelanigripes, Gazelle, Dama—Gazella dama, Gibbon, white-cheeked—Nomascusleucogenys, Goat, Nigerian dwarf—Capra hircus hircus nigerian dwarf,Goat, Nubian—Capra hircus hircus anglo nubian, Goat, San ClementeIsland—Capra hircus hircus san clemente, Gorilla, westernlowland—Gorilla gorilla gorilla, Hog, Ossabaw Island—Sus scrofa scrofa,Horse, Przewalski's—Equus caballus przewalskii or Equus ferusprzewalskii, Hyrax, rock—Procavia capensis, Lemur, ring-tailed—Lemurcatta, Lemur, red-fronted (subspecies of brown lemur)—Lemur fulvusrufus, Lemur, red-ruffed—Varecia variegata rubra, Lion, African—Pantheraleo leo, Macaque, Celebes crested (formerly, Sulawesi)—Macaca nigra,Macaque, liontail—Macaca silenus, Marmoset, Geoffroy's (White-fronted ortufted-eared marmoset)—Callithrix geoffroyi, Meerkat—Suricata suricatta,Mole rat, Damaraland—Cryptomys damarensis, Mole-rat,Naked—Heterocephalus glaber, Monkey, black howler—Alouatta caraya,Mongoose, dwarf—Helogale parvula, Onager, Persian—Equus hemionus onager(SCBI Front Royal only), Orangutan, Bornean—Pongo pygmaeus, Orangutan,Sumatran-Bornean—Pongo pygmaeus abelii, Oryx, scimitar-horned—Oryxdammah, Otter, Asian small-clawed Aonyx cinerea, Otter, North Americanriver—Lontra (formerly, Lutra) canadensis, Panda, giant—Ailuropodamelanoleuca, Panda, red—Ailurus fulgens, Peccary, collared—Tayassutajacu, Porcupine, prehensile-tailed—Coendou prehensilis, Prairie dog,black-tailed—Cynomys ludovicianus, Rabbit, silver fox—Oryctolaguscuniculus, Saki, Guianan (Pale-headed saki)—Pithecia pithecia, Seal,gray—Halichoerus grypus, Siamang—Hylobates syndactylus, Sloth,two-toed—Choloepus didactylus, Squirrel, Prevost's—Callosciurusprevosti, Tamarin, golden lion—Leontopithecus rosalia, Tamarin,golden-headed lion—Leontopithecus chrysomelas, Tenrec, greaterMadagascar—Setifer setosus, Tenrec, small Madagascar hedgehog—Echinopstelfairi, Tiger, Sumatran—Panthera tigris sumatrae, Titi, dusky(Orabussu titi)—Callicebus moloch, Tree shrew, northern—Tupaiabelangeri, Wolf, maned—Chrysocyon brachyurus, and Zebra, Grevy's—Equusgrevyi, pets, farm animals, and wild animals may be treated as well ashumans.

Kits are typically a divided or undivided container that packages orcontains multiple components. The components may be separated or mixed.They may agents and/or delivery devices and/or instructions. They mayinclude mixing vessels or devices.

Spores of anaerobic Clostridium novyi may also be used in combinationwith the other agents described here. The addition of Clostridium sporesachieves any even higher level of survival in a tumor-bearingindividual, even though alone it is less effective than the othertreatments. Amount of spores administered can be, for example, from 10⁵to 10⁹, from 10⁶ to 10⁸, or from 1×10⁷ to 10⁸ per mouse. In oneembodiment the amount of spores is 5.0×10⁷ per mouse. Balb/c miceaverage about 20-30 g. The amount of spores can be scaled according tobody weight of the recipient.

While the precise mechanism of action of the spores is not yet known, itis known that C. novyi-NT infection can elicit a CD8+ T cell-mediatedadaptive immune response specifically against the tumors C. novyi-NT hasinfected. Moreover, it is known that CD8⁺ T cells from C. novyi-NT-curedmice can confer adoptive immunity in a tumor-specific fashion. Thus CD8⁺T cells are involved in the beneficial effect. See Agrawal et al., ProcNatl Acad Sci USA. 2004 Oct. 19; 101(42): 15172-15177. One hypothesis isthat a robust inflammatory response induced by bacterial infectionenhances the adaptive immune response in general (against both bacteriaand tumor cells).

While applicants do not wish to be bound by any theory regarding themechanism of action, it is reasonable to postulate that in order togenerate a potent anti-tumor immune response, two important componentsshould be in place: (a) strong tumor antigens or a robust inflammatoryresponse to enhance the adaptive anti-tumor immune response if strongtumor antigens are not present (as can be provided by C. novyi-NTinfection); and (b) cytotoxic T cells that are not inactivated by theimmune checkpoints (as can be provided by anti-PD-1/anti-CTLA/4 antibodytreatment) or MDSC (as can be provided by entinostat/5-azacitidine).

A recent clinical study demonstrated that epigenetic modulation exertedmajor therapeutic effects on a small fraction of patients with non-smallcell lung cancer (NSCLC) (36). Other studies have suggested that5-azacitidine up-regulates genes and pathways related to both innate andadaptive immunity and genes related to immune evasion in NSCLC lines(35). These important studies as well as the recent clinical trials withimmune check point blockade have led to the initiation of a clinicaltrial combining PD-1 antibody, 5-azacitidine and entinostat in NSCLCpatients(http://clinicaltrialsgov/ct2/show/NCTO1928576?term=entinostat+pd-1&rank=1).It will be interesting to determine the importance of both changes ingene expression in the tumor cells and changes in the number andfunction of MDSCs in this trial. Our observations raised a number ofquestions. For example, what are the mechanisms underlying the selectivesuppression of MDSCs by epigenetic and PI3K inhibitors? Would otherapproaches (e.g. myelosuppressive agents) targeting immune suppressorcells synergize with immune checkpoint blockade for complete eradicationof solid tumors and their metastases? Would priming with epigeneticinhibitors prior to immune checkpoint blockade work as well asconcomitant administration of the two as done in the current study?Experiments addressing these questions may lead to the development ofmore effective therapies harnessing the power of immunity.

The above disclosure generally describes the present invention. Allreferences disclosed herein are expressly incorporated by reference. Amore complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only, and are not intended to limit the scope of theinvention.

Example 1 Materials and Methods

-   -   Reagents. HyClone RPMI 1640 with L-Glutamine and McCoy's 5A were        purchased from Invitrogen Life Technologies. HyClone Fetal        Bovine Serum (FBS) was purchased from Thermo Scientific.        Collagenase from Clostridium histolyticum, Type IV was purchased        from Sigma-Aldrich. The following antibodies and reagents were        used for animal experiments: mCD152 (mCTLA-4) monoclonal        antibody (9H10, BioXCell), mPD-1 monoclonal antibody (RMP1-14,        BioXCell), mCD25 monoclonal antibody (PC61.5.3, BioXCell), mLy6G        monoclonal antibody (RB6-8C5, BioXCell), Polyclonal Hampster IgG        (BioXCell), entinostat (BPS Bioscience), 5-azacytidine        (Invivogen).

Cell Lines. 4T1 (CRL-2539, murine breast tumor cells) and CT26(CRL-2638, murine colorectal adenocarcinoma) were purchased from ATCC.Both tumor cell lines were grown in McCoy's 5A supplemented with 10%Fetal Bovine Serum at 37° C., 5% CO2.

Preparation of Illumina Genomic DNA Libraries. Genomic DNA librarieswere prepared following Illumina's (Illumina) suggested protocol withthe following modifications. 2-3 μg of genomic DNA were diluted with TEto a final volume of 100 μl and sheared in a Covaris sonicator (Covaris)to an average size of 200 bp. DNA was then purified with a Nucleospinkit (Macherey-Nagel), and eluted with 50 μl of elution buffer. Allreagents used for the following steps were from New England Biolabs(NEB) unless otherwise noted. 45 μl of purified DNA was then mixed with40 μl of ddH2O, 10 μl of End Repair buffer and 5 μl of End Repairenzyme. The mixture was incubated at 20° C. for 30 min, purified by aQiagen PCR purification kit (Qiagen) and eluted with 42 μl of elutionbuffer (EB) warmed to 70° C. The end repair reaction was then A-tailedusing 42 μl of end-repaired DNA, 5 μl of 10×dA Tailing Reaction bufferand 5 μl of Klenow Fragment (3′ to 5′ exo-) and incubated at 37° C. for30 min then purified with a MinElute PCR purification kit (Qiagen).Purified DNA was eluted with 27 μl of 65oC EB. Adaptor ligation wasperformed with 25 μl of A-tailed DNA, 10 μl of PE-adaptor (Illumina), 10μl of 5× Ligation buffer and 5 μl of Quick T4 Ligase. The ligationmixture was incubated at 20° C. for 15 min. Purification was done usingby mixing 50 μl of the ligation mixture with 200 μl of NT buffer fromNucleoSpin Extract II kit (Clontech) and loaded into a NucleoSpincolumn. The column was centrifuged at 14,000 g in a desktop centrifugefor 1 min, washed once with 600 μl of wash buffer (NT3 from Clontech),and centrifuged again for 2 min to dry completely. DNA was eluted in 50μl elution buffer included in the kit. Purified ligated DNA was PCRamplified under the following conditions. Around 10 reactions were setup consisting of 32.5 μl of H2O, 2.5 μl (DMSO), 10 μl of 5× Phusion HFbuffer, 1.0 μl of a dNTP mix containing 10 mM of each dNTP, 0.5 μl ofIllumina PE primer #1, 0.5 μl of Illumina PE primer #2, 0.5 μl ofHotstart Phusion polymerase, and 5 μl of ligated DNA. The PCR programused was: 98° C. 1 minute; 10 to 16 cycles of 98° C. for 20 seconds, 65°C. for 30 seconds, 72° C. for 30 seconds; and 72° C. for 5 min. Thereactions were then pooled and purified with a 1:2 mixture of PCRproduct and NT buffer from a NucleoSpin Extract II kit and purified viathe protocol contained within the kit. Library DNA was eluted with 70°C. elution the DNA concentration was estimated by absorption at 260 nmwith a nanodrop and then the samples proceeded to Sureselect exomeisolation.

Exome Capture. The mouse exome was captured following a protocol fromAgilent's SureSelect Paired-End Mouse Exome Kit (Agilent) with thefollowing modifications. (1) A hybridization mixture was preparedcontaining 25 μl of SureSelect Hyb #1, 1 μl of SureSelect Hyb #2, 10 μlof SureSelect Hyb #3, and 13 μl of SureSelect Hyb #4. (2) 3.4 μl (0.5μg) of the PE-library DNA described above, 2.5 μl of SureSelect Block#1, 2.5 μl of SureSelect Block #2 and 0.6 μl of Block #3; was loadedinto one well in a 384-well Diamond PCR plate (cat #AB-1111,Thermo-Scientific), sealed with 2 layers of microAmp clear adhesive film(cat #4306311; ABI) and placed in GeneAmp PCR system 9700 thermocycler(Life Sciences Inc.) for 5 min at 95° C., then held at 65° C. (with theheated lid on). (3) 25 μl of hybridization buffer from step (1) washeated for at least 5 min at 65° C. in another sealed plate with heatedlid on. (4) 5 μl of SureSelect Oligo Capture Library, 1 μl ofnuclease-free water, and 1 μl of diluted RNase Block (A 1:1 RNase Block,nuclease-free water mix) were mixed and heated at 65° C. for 2 min inanother sealed 384-well plate. (5) While keeping all reactions at 65°C., 13 μl of Hybridization Buffer from Step (3) was quickly added to the7 μl of the SureSelect Capture Library Mix from Step (4) and then theentire contents (9 μl) of the library from Step (2). The mixture waspipetted up and down 10 times. (6) The 384-well plate was sealed tightlyand the hybridization mixture was incubated for 24 hours at 65° C. witha heated lid. After hybridization, five steps were performed to recoverand amplify the captured DNA library: (1) 50 μl of Dynal MyOneStreptavidin Cl magnetic beads (Cat #650.02, Invitrogen Dynal) wasplaced in a 1.5 ml microfuge tube and vigorously resuspended on a vortexmixer. Beads were washed three times by adding 200 μl of SureSelectBinding buffer, mixing on a vortexer for five seconds and then placingthe tubes in a Dynal magnetic separator prior to removing thesupernatant after. After the third wash, beads were resuspended in 200μl of SureSelect Binding buffer. (2) To bind captured DNA, the entirehybridization mixture described above (29 μl) was transferred directlyfrom the thermocycler to the bead solution and immediately inverted a 4times to mix; the hybridization mix/bead solution was then incubated inan Eppendorf thermomixer at 850 rpm for 30 min at room temperature. (3)To wash the beads, the supernatant was removed from beads after applyinga Dynal magnetic separator and the beads were resuspended in 500 μlSureSelect Wash Buffer #1 by mixing on vortex mixer for 4 seconds andincubated for 15 min at room temperature. Wash Buffer #1 was thenremoved from beads after magnetic separation. The beads were furtherwashed three times, each with 500 μl pre-warmed SureSelect Wash Buffer#2 after incubation at 65° C. for 10 min. After the final wash,SureSelect Wash Buffer #2 was completely removed. (4) To elute capturedDNA, the beads were suspended in 50 μl SureSelect Elution Buffer,vortex-mixed and incubated for 10 min at room temperature. Thesupernatant was removed after magnetic separation, collected in a new1.5 ml microcentrifuge tube, and mixed with 50 μl of SureSelectNeutralization Buffer. DNA was purified with a Qiagen MinElute columnand eluted in 17 μl of 65° C. buffer EB to obtain 15 μl of captured DNAlibrary. (5) The captured DNA library was amplified in the followingway: Approximately 15 PCR reactions each containing 9.5 μl of H2O, 3 μlof 5×Phusion HF buffer, 0.3 μl of 10 mM dNTP, 0.75 μl of DMSO, 0.15 μlof Illumina PE primer #1, 0.15 μl of Illumina PE primer #2, 0.15 μl ofHotstart Phusion polymerase, and 1 μl of captured exome library were setup. The PCR program used was: 98° C. for 30 seconds; 14 cycles of 98° C.for 10 seconds, 65° C. for 30 seconds, 72° C. for 30 seconds; and 72° C.for 5 min. To purify PCR products, 225 μl of PCR mixture (from 15 PCRreactions) was mixed with 450 μl of NT buffer from NucleoSpin Extract IIkit and purified as described above. The final library DNA was elutedwith 30 μl of 65° C. elution buffer and DNA concentration was estimatedby OD₂₆₀ measurement.

Preparation of cDNA for Sequencing Analysis. mRNA was prepared from 5-10μg total RNA using two rounds of poly-A selection using Dynal oligo-dTmagnetic beads following the manufacturers protocol (Life Technologies)and eluted the second time with 13 μl of elution buffer. Double-stranded(ds) cDNA was prepared using the SuperScript ds-cDNA kit (Invitrogen)with the following modifications. 12 μl of the isolated mRNA was addedto 2 μl of 50 ng/μl random hexamers and incubated at 70° C. for 10minutes then placed on ice. 4.4 μl of 5× First Strand Buffer, 2.2 μl of0.1 M DTT and 1.1 μl of a 10 mM dNTP mixture were added to the tube andincubated at 45° C. for 2 minutes upon which 1.5 μl of SSII enzyme wasadded and the entire mixture incubated for an additional 1 hour and thenplaced on ice. Second Strand cDNA was made by adding to the first strandreaction 90 μl of ddH₂O, 30 μl of 5× Second Strand Buffer, 3 μl of 10 mMdNTPs, 4 μl of DNA Pol I, 1 μl of RNaseH and 1 μl of E. coli DNA ligase.The mixture was then incubated at 16° C. for 2 hours upon which 2 μl ofT4 ligase was added and incubated for an additional 5 minutes. Theresulting cDNA was then purified using a Qiagen PCR purification kitfollowing the manufacturer's instructions and eluted twice with 50 μl of70° C. elution buffer each time. The 100 μl of cDNA was used for theconstruction of illumina libraries following the genomic DNA libraryprotocol with the cDNA in place of genomic DNA.

Somatic Mutation Identification. Libraries were sequenced on IlluminaGAIIx or HiSeq Genome Analyzer. Sequencing reads were analyzed andaligned to mouse genome mm9 with CASAVA (Illumina). A mismatched basewas identified as a mutation only when (i) it was identified by four ormore distinct pairs containing at least 2 reads in the forward and 2 inthe reverse orientation; (ii) the number of distinct tags containing aparticular mismatched base was at least 30% of the total distinct tags.

Identification of Putative Expressed H2-(d) Epitopes. Identified somaticmutations were cross-referenced against the RNAseq data to determinewhich mutations were expressed. The amino acid change corresponding tothe positive mutations were then used for mutant epitope identificationby isolating 8 amino acids up- and down-stream of the mutant residue.This seventeen amino acid sequence was than processed using the netMHCepitope identification algorithm (netMHC v3.4) for potential 9 aminoacid binders to H2-k(d), H2-L(d) and H2-D(d). The cutoff used was 500 nMaffinity or higher which corresponds to moderate to high affinitybinders. Genes were determined to be expressed if the normalized counts(normalized to length per million reads) of RNAseq data were greaterthan 0.5.

Animal Models. Animal research was approved and overseen by JohnsHopkins University Institutional Animal Care and Use Committee. Six toeight weeks old female BALB/C mice (Harlan Laboratories) were used forall animal experiments. 5×10⁶ 4 T1 tumor cells or 5×10⁶ CT26 tumor cellswere inoculated subcutaneously into the right flank of each mouse.Tumors were allowed to grow for 11 days prior to randomization andtreatment. CT26 bearing mice were given 10 mg/kg of anti-PD-1 and/oranti-CTLA-4 antibodies intraperitoneally on day 11, 13, 15, 17, 20, 23and 26 post-tumor implantations and 4T1 bearing mice were given 10 mg/kganti-PD-1 and/or anti-CTLA-4 antibodies intraperitoneally on day 11, 13,15, and 17 post-tumor implantations. 4T1 tumor bearing mice were given asingle dose of 10 mg/kg anti-CD25 antibody or 10 mg/kg anti-Ly6Gantibody intraperitoneally on day 12 post-tumor implantation for celldepletion studies. All antibodies were diluted to appropriateconcentrations in 100 μl sterile PBS, pH 7.4 (Invitrogen LifeTechnologies). Entinostat and 5-azacytidine treatments were started onday 12 post-tumor implantation at a dose of 20 mg/kg and 0.8 mg/kg,respectively. 4T1 bearing mice were injected intraperitoneally on day12, 14, 16, and 18. J32 was given at 22 mg/kg by intraperitonealinjections on day 12, 14, 16, and 18 of 4T1 tumor implantation. Tumorswere measured for 30 days from the start of the treatment at indicatedintervals in the results section. Tumor volume was calculated aslength×width²×0.5.

Metastasis Analysis. On day 46 post-tumor implantations, 4T1 tumorbearing mice were euthanized according to the IACUC guidelines. Lungs,livers and spleens were harvested and fixed in 10% Neutral BufferedFormalin Solution (Sigma-Aldrich) and metastatic nodules were countedfrom at least three mice per group.

Flow Cytometry. The following antibodies and reagents were used for flowcytometry: CD16/32 (BD Biosciences), CD3e Alexa Fluor 488 (14-C11; BDBiosciences), CD4 Brilliant Violet 421 (GK1.5, BD Biosciences), CD8aPerCP-Cy5.5 (53-6.7, BD Biosciences), CD25 PE (PC61, BD Biosciences),Foxp3 Alexa Fluor 647 (MF23, BD Biosciences), CD11b Alexa Fluor 700(M1/70, BD Biosciences), I-A/I-E Alexa Fluor 488 (M5/114.15.2, BDBiosciences), Ly-6C PerCP-Cy5.5 (AL-21, BD Biosciences), CD11c PE (HL3,BD Biosciences), F4/80 APC (BM8, Biolegend), Ly-6G Pacific Blue (1A8,Biolegend), CD45 Pacific Orange (30-F11, Invitrogen Life Technologies),and Live/Dead Fixable Near IR Dead Cell Stain (Invitrogen LifeTechnologies). Flow cytometry was performed with LSR II (BD Biosciences)and the data was analyzed with FACS Diva software (BD Biosciences). Toassess the level of circulating G-MDSC population, blood samples werecollected from the mice 7 days after the initiation of theanti-PD-1/anti-CTLA-4 antibody treatments with or without5-AZA/Entinostat. 150 μl of blood was collected into K₂EDTA BDMicrotainer (BD Biosciences) from either right or left facial vein. RBCsfrom anti-coagulated blood samples were immediately lysed using 2 ml of1×BD FACS Lyse (BD Biosciences) for 3 minutes and the samples werewashed twice in ice-cold BD FACS Buffer (BD Biosciences). After 5-minuteincubation with Live/Dead Fixable Near IR Dead Cell Stain and two washeswith ice-cold BD FACS Buffer, the samples were stained with appropriateantibodies. For analysis, we used previously established phenotypiccriteria of these cells as CD45⁺CD11b⁺Ly6G⁺Ly6C^(lo) F4/80⁻MHCII⁻ cellsand total CD45 positive cells were used as a common denominator. Toassess the level of intratumoral CD8⁺ and regulatory T cell populations,lymphocytes were first purified from tumor samples excised from mice 7days after the initiation of the anti-PD-1/anti-CTLA-4 antibodytreatments with or without 5-AZA/Entinostat. Briefly, primary tumortissues were harvested, weighed, and minced to fine fragments.Collagenase IV (Sigma-Aldrich) at 1 mg/ml in HBSS (Invitrogen LifeTechnologies) was added to each sample at a ratio of 1 ml per 200 mg oftumor tissue. Samples were incubated on an end-over-end shaker for 30minutes at 37° C. The resulting tissue homogenates were 0.4 μm filtered,washed three times in ice-cold BD FACS Buffer (BD Biosciences), and5×10⁶ cells per sample were used for antibody labelling. CD8⁺ T celllevel was assessed using previously established phenotypic criteria ofCD45⁺CD3⁺CD8⁺ and total CD45⁺CD3⁺ cells was used as a commondenominator. Treg cell level was assessed using previously establishedphenotypic criteria of CD45⁺CD3⁺CD4⁺CD25⁺FoxP3⁺ and total CD45⁺CD3⁺CD4⁺cells were used as a common denominator.

Cell Isolation. MDSCs from 4T1 tumor-bearing animals were isolated fromspleens using Myeloid-Derived Suppressor Cell Isolation Kit, mouse(Miltenyi Biotec) and BD FACS Aria III cell sorter (BD Biosciences).CD8⁺ cells were isolated from spleens of 4T1-bearing animals treatedwith anti-PD-1 and anti-CTLA-4 antibodies using CD8a⁺ T Cell IsolationKit II, mouse (Miltenyi Biotec) and BD FACS Aria III cell sorter (BDBiosciences). MDSC, Tregs, and CD8⁺ T cells were isolated and culturedby following the manufacturer's protocols and published protocols (38,39). The purity of G-MDSCs (CD11b⁺Ly6G⁺Ly6C^(lo) F4/80⁻MHC-II⁻) and CD8⁺(CD3⁺CD8⁺) populations were greater than 95% as determined by flowcytometry, and the viability was greater than 95% for these populationsas determined by trypan blue staining.

In Vitro Survival Assay. MDSCs and CD8⁺ T cells were plated on 96-wellplate at 2×10⁶ cells/ml in RPMI1640 medium supplemented with 10% FBS.CD8⁺ T cell cultures were supplemented with recombinant interleukin-2(Invitrogen Life Technologies) at 2000 U/ml. 4T1 cells in McCoy 5Asupplemented with 10% FBS were plated on 96-well plate and cultureduntil they reached >70% confluency. Cells were cultured with entinostat,5-azacytidine, or J32 at concentrations ranging from 0 μM to 50 μM for24 hours at 37° C., 5% CO2. The proportion of viable cells was assessedby incubating the cells with 10% (v/v) Cell Proliferation Reagent WST-1(Roche Applied Science) for 3 hours at 37° C. and measuring OD₄₅₀absorbance of the resulting formazan products.

IFN-γ Assay. Freshly isolated MDSCs and CD8⁺ T cells from 4T1tumor-bearing animals were cultured at MDSC to CD8⁺ T cell ratios of5:1, 2:1, 1:1, 1:2, and 1:5 in presence of recombinant IL-2 (InvitrogenLife Technologies) at 2000 U/mL and CD3/CD28 antibody coated beads(Miltenyi). MDSC and CD8⁺ T cells were cultured at 1:1 ratio withentinostat at concentrations ranging from 0 μM to 0.25 μM. Cell-freesupernatants were collected after 24-hour incubation at 37° C. and IFN-γlevels were assayed using Mouse IFN-γ DuoSet Elisa Development Kit (R&DSystems) according to the manufacturer's instruction.

MDSC Depletion and Adoptive Transfer. For in-vivo depletion of MDSC, 4T1tumor-bearing mice were treated with a single bolus of mLy6G monoclonalantibody at 10 mg/kg administered intraperitoneally on day 11 post-tumorimplantation. For adoptive transfer of MDSC, spleens from 4T1-bearingmice were collected and MDSCs were purified with Myeloid-DerivedSuppressor Cell Isolation Kit, mouse (Miltenyi Biotec). After twosequential column purifications, cells were washed twice in ice-cold1×PBS (Invitrogen Life Technologies) and cell concentration was adjustedto 1×10⁸ cells/ml. Cell viability was greater than 95% as verified withtrypan blue staining and cell purity was greater than 90% as determinedby flow cytometry. Immediately following the isolation, 100 μl of MDSCsat 1×10⁸ cell/ml were administered via tail vein injection on day 11,13, and 15 post 4T1 tumor implantation.

Immunofluorescence. Mice were euthanized according to the JHU IUCACguidelines and primary tumors from 4T1 tumor-bearing mice were excisedusing sterile disposable surgical scalpels (Bard-Parker). Excisedtissues were placed in base molds filled with Tissue-Tek CRYO-OCT(Andwin Scientific) and stored at −80° C. until use. Frozen tissues weresectioned using Leica CM3050 S Cryostat (Leica Biosystems) and tissueswere fixed with 4% Paraformaldehyde (Alfa Aesar), 0.3% Triton X-100(Sigma-Aldrich) in 1×PBS (Invitrogen Life Technologies) for 10 minutes.Slides were washed three times with 0.05% Tween-20 (Sigma-Aldrich) in1×PBS followed by three 5-minute washes with 0.05% Tween-20 in 1×PBS.Tissues were blocked with 3% BSA (Sigma-Aldrich), 0.05% Tween-20 in1×PBS for 30 minutes, followed by an additional 30-minute block with 10%normal goat serum (Invitrogen Life Technologies). Blocked tissues wereincubated overnight in anti-CD8 (YTS 169.4, Abcam) or anti-Ly6G(RB6-8C5, Abcam) at concentrations 1:50, 1:100, and 1:200 at 4° C.Following the overnight staining with primary antibodies, the slideswere washed three times with 0.05% Tween-20 in 1×PBS and incubated with1:500 goat anti-rat AF488 (Invitrogen Life Technologies) or goatanti-rat AF594 (Invitrogen Life Technologies) secondary antibodies for 1hour at 20° C. Slides were washed five times with 0.05% Tween-20 in1×PBS, one drop of Gold/DAPI (Invitrogen Life Technologies) was added tothe tissue samples prior to placing a cover slip, and the slides werestored at 4° C. in dark. For imaging, Nikon Cl Laser Scanning ConfocalSystem, which includes ECLIPSE TE2000-E microscope and EZ-LIMO for NikonCl Confocal v. 2.30 was used.

Reverse-transcription PCR (RT-PCR). 5×10⁶ CT26 or 4T1 cells wereresuspended in 0.75 ml Trizol LS Reagent (Invitrogen Life Technologies)and 0.25 ml chloroform (Sigma-Aldrich). Samples were vortexed for 15seconds and incubated for 10 minutes at 20° C. After a 15-minute 12000 gcentrifugation at 4° C., upper aqueous phase was collected and 0.5 ml of100% isopropanol (Sigma-Aldrich) was added. Samples were centrifuged at12000 g for 10 minutes at 4° C. after 10 minute incubation at 20° C. Theresulting pellets were air dried for less than 10 minutes and wereresuspended with RNAse-free water (Invitrogen Life Technologies) for afinal concentration of 500 ng/μl. PCR was performed using SuperScriptIII One-Step RT-PCR System with Platinum Taq DNA Polymerase (InvitrogenLife Technologies). Annealing temperature was set at 55° C. for H-2D(d),(32m and TAP1, and set at 60° C. for β-actin. Samples were analyzed on1% agarose gel. The following primers were used for RT-PCR: H-2D(d)Forward 5′-agggcaatgagcagagtttc-3′ (SEQ ID NO: 1), H-2D(d) Reverse5′-CCACGTTTTCAGGTCTTCGT-3′ (SEQ ID NO: 2), β2m Forward5′-ATTCACCCCCACTGAGACTG-3′ (SEQ ID NO: 3), β2m Reverse5′-GCTATTTCTTTCTGCGTGCAT-3′ (SEQ ID NO: 4), TAP1 Forward5′-GAGACATGCTGTGTCGGATG-3′ (SEQ ID NO: 5), TAP1 Reverse5′-TGGTGAGAATGGACATGAGC-3′ (SEQ ID NO: 6), β-actin Forward5′-TTCTTTGCAGCTCCTTCGTTGCCG-3′ (SEQ ID NO: 7), β-actin Reverse5′-TGGATGGCTACGTACATGGCTGGG-3′ (SEQ ID NO: 8).

Statistics. All statistics analyses were performed with Prism 5.0(GraphPad Software, Inc). Primary tumor growth curves were firstanalyzed with two-way ANOVA and individual groups were compared with twotailed Wilcoxon rank-sum test. Kaplan-Meier survival curves wereanalyzed with log-rank test. Statistical significance of metastaticlesions, flow cytometric analyses, and in-vitro assays were assessedwith two tailed Wilcoxon rank-sum test.

Example 2

Genetic analysis. We first sequenced the exomes (24,306 genes) of bothCT26 and 4T1 cells. Eight and 3.5 gigabases of generated sequence weremapped to the genome for CT26 and 4T1, respectively. 83.5% (CT26) and72.3% (4T1) of bases in the targeted regions were covered by at least 10unique reads in tumor DNA. Sequencing of the exomes revealed 683 and 47somatic mutations in CT26 and 4T1, respectively (Dataset S1).

It has been shown that ˜10% of the mutant amino acids created by somaticmutations in human colorectal and breast cancers give rise to epitopesthat are predicted to be recognized by the patient's MHC-I alleles (17).To determine whether this was true for the murine colorectal (CT26) andbreast (4T1) tumors, we mapped the somatically mutated epitopes toBALB/c MHC-I using established algorithms. As such predictions aremeaningful only if the mutant genes are expressed, we determined thetranscriptomes of the two cell lines using RNA-Seq. Three hundred andfourteen of the 683 mutations detected in CT26 occurred in expressedgenes, with 28 mutated epitopes predicted to bind with at least moderateaffinity to H2-(d) MHC-I alleles found in BALB/c mice (Dataset S1 andTable 1). The 4T1 cells harbored 27 mutations in expressed genes withonly one predicted to bind to H2-(d) MHC-I alleles. These data areconsistent with the suggestion that CT26 is more immunogenic than 4T1because the former has more mutant epitopes. It is also consistent withthe observation that human tumors associated with environmental mutagens(such as UV light and cigarette smoke) have more mutations than othertumors (18).

TABLE 1 Mutant MHC-I Epitopes predicted from genome andtranscriptome analyses Affinity Name Epitope (nM) H-2 AlleleCT26 Tumor Cell SEQ ID NO. 9 AE2f8 SGPSYATYL 362 H-2Dd SEQ ID NO. 10Haus6 SYETLKKSL  21 H-2Kd SEQ ID NO. 11 Slc20a1 SYTSYIMAI  28 H-2KdSEQ ID NO. 12 Sel11 RYWTGIGVL  46 H-2Kd SEQ ID NO. 13 Glud1 AYVNAIEKI 49 H-2Kd SEQ ID NO. 14 Noc3l SYIKKLKEL  58 H-2Kd SEQ ID NO. 15 Eml5HYLNDGDAI  66 H-2Kd SEQ ID NO. 16 Gnas KVLAGKSTI  74 H-2Kd SEQ ID NO. 17Mtor HHTMMVQAI 126 H-2Kd SEQ ID NO. 18 Map3k5 AYALNRRNL 131 H-2KdSEQ ID NO. 19 Slc20a1 SYIMAICGM 146 H-2Kd SEQ ID NO. 20 Dhx35 YYMRDVIAI211 H-2Kd SEQ ID NO. 21 Pigo LFLKSPTAL 265 H-2Kd SEQ ID NO. 22 Ptpn13PYFRLEHYL 311 H-2Kd SEQ ID NO. 23 Qsox1 SYLRRLPGL 316 H-2KdSEQ ID NO. 24 Tars12 TYWKGNPEM 332 H-2Kd SEQ ID NO. 25 Anks6 GYEAVVRLL338 H-2Kd SEQ ID NO. 26 Slc41a2 PYLTALDDL 365 H-2Kd SEQ ID NO. 27 PdgfraLFVTVLEVI 404 H-2Kd SEQ ID NO. 28 Rwdd2b VYFTINVNL 432 H-2KdSEQ ID NO. 29 Vps26b SYTEQNVKL 460 H-2Kd SEQ ID NO. 30 Qsox1 FYTSYLRRL496 H-2Kd SEQ ID NO. 31 Phf3 FPPQNMFEF   5 H-2Ld SEQ ID NO. 32 Trim26SPEAQLFAV 112 H-2Ld SEQ ID NO. 33 Zfp449 EPQIAMDDM 115 H-2LdSEQ ID NO. 34 Csnk2b IPDEAMVKL 184 H-2Ld SEQ ID NO. 35 Zeb1 EPQVEPLDF272 H-2Ld SEQ ID NO. 36 Ttc15 DPFATPLSM 485 H-2Ld 4T1 Tumor CellSEQ ID NO. 37 Qars FPPDAINNF  27 H-2Dd

Example 3

Effects of immune checkpoint blockade. We then tested the effects ofimmune checkpoint blocking antibodies on tumors derived from these cellsin mice. BALB/c mice bearing subcutaneous CT26 tumors of moderate sizes(˜400 mm³) were used for the initial experiments. While repeatedtreatment with anti-CTLA-4 or anti-PD-1 antibodies as single agentsretarded tumor growth, tumor eradication was not observed (FIGS. 1A andB). Combination therapy with both antibodies resulted in eradication oftumors in vast majority of the mice. Conversely, tumors larger than 600mm³ did not respond to the combined anti-PD-1/anti-CTLA4 treatment aswell (FIG. 1C), with only 4 out of 11 animals showing long-term survival(FIG. 1D).

Next, BALB/c mice with well-established 4T1 tumors (˜400 mm³) wereevaluated; these tumors spontaneously metastasize to the lungs and otherorgans. The 4T1 tumor model is highly recalcitrant to most therapeuticagents, including immunotherapy (16). The animals generally succumb tometastatic disease, even when the primary tumor is surgically removed(19). Small number of the primary tumors showed durable response toantibody treatment. Similar to the mice with large CT26 tumors, only 3out of 10 animals showed complete regression of their primary tumorswhen treated with both anti-PD-1 and anti-CTLA-4 antibodies, and thesewere the only long-term survivors (FIGS. 1 E and F).

Example 4

Epigenetic modulation. We hypothesized that the tumors in the animalsthat had not been cured might have down-regulated the expression ofMHC-I-related genes through epigenetic silencing in tumor cells. Indeed,this hypothesis forms the basis for therapies involving epigeneticmodulation (20), using inhibitors of either DNA methyltransferase orhistone deacetylase (HDAC). To evaluate this possibility, we treatedanimals bearing large CT26 tumors (>600 mm³) as described above withanti-PD-1/anti-CTLA-4 antibodies as well as 5-azacytidine (AZA, a DNAmethyltransferase inhibitor) and entinostat (ENT, a class I HDACinhibitor). The tumors responded to this regimen remarkably well, witheradication of primary tumors in 10 out of 11 mice and 100% survival 60days after tumor implantation (FIG. 1D). Similarly, in response to theanti-PD-1/anti-CTLA-4 plus AZA/ENT treatment, mice with 4T1 tumors (˜400mm³) showed complete regression of all primary tumors three weeks aftertreatment initiation and 80% survival 100 days after tumor implantation(FIGS. 1 E and F). Temporary self-limiting toxicity, as indicated bybody weight changes, was observed when entinostat was used (FIG. 37).However, the addition of anti-PD-1/anti-CTLA-4 antibodies did not add tothe toxicity.

In parallel experiments, we treated 4T1 tumor-bearing mice as describedabove but sacrificed them six weeks after tumor implantation. We thenexamined their primary tumors as well as lungs and other organs formetastasis. The primary tumors were eradicated in all five mice treatedwith anti-PD-1/anti-CTLA-4 antibodies plus AZA/entinostat and none ofthem showed any metastases (FIG. 1G and Table 2). In contrast, all fivemice with anti-PD-1/anti-CTLA-4 treatment alone still had large primarytumors and an average of 11 lung metastases. We also treated thetumor-bearing mice with anti-PD-1/anti-CTLA-4 antibodies plus eitherentinostat or AZA. No primary tumors or metastases were found in any ofthe mice treated with anti-PD-1/anti-CTLA-4 antibodies plus entinostat,suggesting that when combined with PD-1/CTLA-4 double blockade, class IHDAC inhibitors alone (without DNA methylation inhibitors) weresufficient to eradicate both primary tumors and metastasis (FIG. 1G andTable 2). In the mice treated with anti-PD-1/anti-CTLA-4 antibodies plusAZA, the primary tumors were not eradicated, though no metastases wereobserved. Without PD-1/CTLA-4 inhibition, entinostat, AZA, alone or incombination, were unable to eradicate either primary tumor or metastasis(FIG. 1G and Table 2). When PD-1/CTLA-4 inhibition was not applied,metastatic lesions were observed in multiple organs in addition to thosein the lungs.

TABLE 2 4T1 Primary Tumors and Metastatic Lesions Treatment PrimaryTumor Lung Liver Spleen Group Mouse No Vol (mm³) Mets Mets Mets P + C 10 0 0 0 AZA + ENT 2 0 0 0 0 3 0 0 0 0 4 0 0 0 0 5 0 0 0 0 AVERAGE 0 0 00 STDEV 0 0 0 0 P + C 1 0 0 0 0 ENT 2 0 0 0 0 3 0 0 0 0 4 0 0 0 0 5 0 00 0 AVERAGE 0 0 0 0 STDEV 0 0 0 0 P + C 1 435 0 0 0 AZA 2 595 0 0 0 31069 0 0 0 4 754 0 0 0 AVERAGE 713 0 0 0 STDEV 271 0 0 0 P + C 1 2221 120 0 2 1768 14 0 0 3 2456 21 0 0 4 577 0 0 0 5 1874 9 0 0 AVERAGE 1779 110 0 STDEV 726 8 0 0 AZA + ENT 1 2891 34 10 0 2 2855 14 0 0 3 3613 22 492 4 2726 25 0 0 5 3597 22 7 1 AVERAGE 3137 23 13 1 STDEV 432 7 20 1 ENT1 3005 25 10 1 2 5038 20 3 0 3 2275 0 0 0 4 3063 22 6 3 5 4032 20 0 0AVERAGE 3483 17 4 1 STDEV 1070 10 4 1 AZA 1 2345 20 0 0 2 2454 16 43 3 32477 20 0 1 4 4358 17 16 0 5 2117 14 0 0 AVERAGE 2750 17 12 1 STDEV 9103 19 1 Untreated 1 4359 32 3 2 2 3432 28 0 2 3 3185 18 28 0 AVERAGE 365926 10 1 STDEV 619 7 15 1

Example 5

Mechanistic studies. As noted above, we expected that the epigeneticmodulators were increasing the expression of MHC-I-related genes,thereby making the cancer cells more susceptible to killing by T cells.To test this expectation, we analyzed the expression of genes involvedin MHC-I presentation by reverse transcription-polymerase chain reaction(RT-PCR) in CT26 and 4T1 cells treated with AZA, entinostat, or thecombination of the two. Expression of the MHC-I, β-2 microglobulin(B2M), and transporter associated with antigen processing 1 (TAP1) geneswere detected in both tumor cell lines in the absence of treatment.However, exposure to epigenetic modulators did not significantlyincrease the expression (FIG. S2).

We then determined whether the epigenetic modulators affected T cellaccumulation within the tumors. As assessed by flow cytometry,tumor-infiltrating CD8⁺ T cells increased by approximately four-foldafter PD-1/CTLA-4 inhibition (FIGS. 2 A and B). The addition of AZA andentinostat did not increase tumor-infiltrating CD8⁺ T cells further.However, inclusion of AZA and entinostat in the treatment regimenresulted in a significant decrease in tumor-infiltrating FoxP3⁺ Tregscompared to either untreated tumors or tumors treated withanti-PD-1/CTLA-4 antibodies (FIGS. 2 C and D).

We next analyzed MDSCs by flow cytometry, as these myeloid-derivedimmature cells are often elevated in tumor-bearing hosts and have potentimmunosuppressive activities (21, 22). We found that 4T1 tumor-bearingmice had a five to seven-fold increase in circulating Granulocytic MDSCs(G-MDSCs, defined as CD11b⁺Ly6G⁺Ly6C^(lo) MHC-II⁻) compared tonon-tumor-bearing animals (FIG. 2E, FIGS. S3 A and B). Large numbers ofG-MDSCs were also observed in the spleen and tumor (FIG. S3B). Additionof entinostat or AZA/entinostat to PD-1/CTLA-4 inhibition resulted in astriking reduction in the number of circulating G-MDSCs, bringing themdown to a level similar to that observed in non-tumor-bearing mice(FIGS. 2 E and F). Interestingly, the epigenetic modulators alone or AZAplus anti-PD-1/anti-CTLA-4 antibodies failed to abate the G-MDSCs. Theepigenetic modulators substantially reduced the number oftumor-infiltrating G-MDSCs as well when combined with immune checkpointblockade (FIGS. 2 G and H).

These data are consistent with the hypothesis that immune checkpointblockade leads to expansion of cytotoxic effector T cells (Teffs), butthe Teffs are not fully functional unless immune suppressor cells arereduced by treatment with epigenetic modulators. To further test thishypothesis, we used neutralizing antibodies against CD25 or Ly6G todeplete Tregs or G-MDSCs, respectively, in mice bearing 4T1 tumors(23-25). We found that anti-Ly6G, when used in combination withanti-PD-1/anti-CTLA-4 antibodies, were as effective as the epigeneticmodulators (FIG. 3A). Flow cytometry showed a substantial reduction inG-MDSC levels after anti-Ly6G treatment (FIG. 3B). In contrast,anti-CD25 treatment only showed marginal improvement in efficacy whencombined with immune checkpoint blockade (FIG. 3A). However, it shouldbe noted that the anti-CD25 treatment may also affect activated Teffs,which can transiently express CD25. As expected, without immunecheckpoint blockade, both anti-CD25 and anti-Ly6G were ineffective (FIG.3A).

To evaluate directly the ability of the tumor-induced G-MDSCs tointerfere with the effects of immune checkpoint blockade, we isolatedthem from 4T1 tumor-bearing mice by affinity purification. We theninjected the purified G-MDSCs into 4T1 tumor-bearing mice treated withanti-PD-1/anti-CTLA-4 antibodies plus AZA/entinostat. The adoptivetransfer of G-MDSCs significantly attenuated the response to thecombination therapy (FIG. 3C). Based on above results, we concluded thatthe effects of epigenetic modulation were more likely the result ofdepletion of G-MDSCs than of direct depletion of Tregs.

To investigate whether epigenetic modulation directly affected G-MDSCs,we purified these cells from 4T1 tumor-bearing mice as described aboveand treated them with entinostat or AZA in vitro. G-MDSCs showedmarkedly reduced viability after entinostat treatment in adose-dependent fashion (FIG. 4A). Conversely, AZA had no effect atcomparable concentrations (FIG. 4B). We also treated 4T1 tumor cellswith the same concentrations of entinostat or AZA and found themunresponsive (FIGS. 4 A and B). Importantly, entinostat had only modesteffects on CD8⁺ T cells (FIG. 4A), creating a large therapeutic windowin which G-MDSCs can be depleted while sparing CD8⁺ T cells. Finally, weco-cultured CD8⁺ T cells with G-MDSCs and analyzed the concentration ofinterferon-y (IFN-γ) in culture medium by enzyme-linked immunosorbentassay (ELISA) following T cell activation with CD3 and CD28 antibodies.G-MDSCs inhibited IFN-γ secretion (FIG. 4C), whereas inclusion ofentinostat in the culture medium reversed the inhibition in adose-dependent manner (FIG. 4D). These data supported the notion thatG-MDSCs directly inhibit the function of CD8⁺ T cells and entinostatalleviates the inhibition by directly suppressing G-MDSCs.

To further confirm this conclusion, as well as to provide additionaltherapeutic approaches to achieve the same goal, we searched for othertherapeutic agents that might suppress G-MDSC function.Phosphatidylinositide 3-kinases (PI3Ks) are known to play importantroles in hematopoietic cell biology and can activate Gr1⁺/CD11b⁺ myeloidcells (26). We had previously developed a diverse array of PI3Kinhibitors and chose to test one (J32) with high cellular potency(27-29). J32 proved to be cytotoxic to G-MDSCs at nanomolarconcentrations (EC₅₀ of 14.3 nM) and much less toxic to CD8⁺ T cells(EC₅₀ of 94.6 nM) (FIG. S4A). Treatment of 4T1 tumor-bearing mice with arelatively low dose of J32 (22 mg/kg) in combination withanti-PD-1/anti-CTLA-4 antibodies resulted in a marked reduction incirculating G-MDSCs (FIG. S4B) and eradication of 4T1 tumors in 80% ofthe animals (FIG. S4C). Alone, J32 had no appreciable effect on the 4T1tumor growth.

Example 5

4T1 tumor cells were subcutaneously injected into BALB/c mice. On day10, 12, 14 and 16 after the tumor cell injection, anti-PD-1 (10 mg/kg)and anti-CTLA-4 (10 mg/kg) antibodies were injected intraperitoneallyinto the mice in groups 2, 3, 4 and 5. On day 11, 13, 15 and 17,entinostat (ENT, 20 mg/kg) and 5-azacytidine (AZA, 0.8 mg/kg) wereinjected intraperitoneally into the mice in groups 4 and 5. On day 13and 15, C. novyi-NT spores (50 million per mouse) were injected directlyinto the subcutaneous 4T1 tumors on mice in groups 1, 3 and 5. Mousesurvival was then followed closely until day 100 after tumor cellinjection. Survival curves are shown in FIG. 9. The dead mice dissectedfor pathological assessment invariably had extensive lung metastases.

The efficacy for checkpoint blockade (anti-PD-1/anti-CTLA-4) plusepigenetic inhibition (ENT/AZA) shown in this experiment (50% cure rate)was somewhat lower than that shown in the prior examples (80% curerate). However, the efficacy for anti-PD-1/anti-CTLA-4 alone in thisexperiment was lower as well (10% cure rate here versus 30% in the priorexamples). The efficacy enhancement observed in the combination therapyhas not been diminished. The variation in efficacy is likely due to thequality of the antibodies from different manufacturing lots.

REFERENCES

The disclosure of each reference cited is expressly incorporated herein.

-   1. Korman A J, Peggs K S, & Allison J P (2006) Checkpoint blockade    in cancer immunotherapy. Advances in immunology 90:297-339.-   2. Pentcheva-Hoang T, Corse E, & Allison J P (2009) Negative    regulators of T-cell activation: potential targets for therapeutic    intervention in cancer, autoimmune disease, and persistent    infections. Immunol Rev 229(1):67-87.-   3. Pardoll D M (2012) The blockade of immune checkpoints in cancer    immunotherapy. Nat Rev Cancer 12(4):252-264.-   4. Nagaraj S, Youn J I, & Gabrilovich D I (2013) Reciprocal    relationship between myeloid-derived suppressor cells and T cells. J    Immunol 191(1):17-23.-   5. Chen L & Flies D B (2013) Molecular mechanisms of T cell    co-stimulation and co-inhibition. Nature reviews. Immunology    13(4):227-242.-   6. Talmadge J E & Gabrilovich D I (2013) History of myeloid-derived    suppressor cells. Nat Rev Cancer 13(10):739-752.-   7. Lippitz B E (2013) Cytokine patterns in patients with cancer: a    systematic review. Lancet Oncol 14(6):e218-228.-   8. Zou W (2006) Regulatory T cells, tumour immunity and    immunotherapy. Nature reviews. Immunology 6(4):295-307.-   9. Hodi F S, et al. (2010) Improved survival with ipilimumab in    patients with metastatic melanoma. N Engl J Med 363(8):711-723.-   10. Topalian S L, et al. (2012) Safety, activity, and immune    correlates of anti-PD-1 antibody in cancer. N Engl J Med    366(26):2443-2454.-   11. Brahmer J R, et al. (2012) Safety and activity of anti-P D-L1    antibody in patients with advanced cancer. N Engl J Med    366(26):2455-2465.-   12. Wolchok J D, et al. (2013) Nivolumab plus ipilimumab in advanced    melanoma. N Engl J Med 369(2):122-133.-   13. Corbett T H, Griswold D P, Jr., Roberts B J, Peckham J C, &    Schabel F M, Jr. (1975) Tumor induction relationships in development    of transplantable cancers of the colon in mice for chemotherapy    assays, with a note on carcinogen structure. Cancer Res    35(9):2434-2439.-   14. Belnap L P, Cleveland P H, Colmerauer M E, Barone R M, & Pilch Y    H (1979) Immunogenicity of chemically induced murine colon cancers.    Cancer Res 39(4):1174-1179.-   15. Dexter D L, et al. (1978) Heterogeneity of tumor cells from a    single mouse mammary tumor. Cancer Res 38(10):3174-3181.-   16. Pulaski B A & Ostrand-Rosenberg S (1998) Reduction of    established spontaneous mammary carcinoma metastases following    immunotherapy with major histocompatibility complex class II and    B7.1 cell-based tumor vaccines. Cancer Res 58(7):1486-1493.-   17. Segal N H, et al. (2008) Epitope landscape in breast and    colorectal cancer. Cancer Res 68(3):889-892.-   18. Vogelstein B, et al. (2013) Cancer genome landscapes. Science    339(6127):1546-1558.-   19. Rashid O M, et al. (2013) Resection of the primary tumor    improves survival in metastatic breast cancer by reducing overall    tumor burden. Surgery 153(6):771-778.-   20. Lampen M H & van Hall T (2011) Strategies to counteract MHC-I    defects in tumors. Current opinion in immunology 23(2):293-298.-   21. Ostrand-Rosenberg S & Sinha P (2009) Myeloid-derived suppressor    cells: linking inflammation and cancer. J Immunol 182(8):4499-4506.-   22. Gabrilovich D I, Ostrand-Rosenberg S, & Bronte V (2012)    Coordinated regulation of myeloid cells by tumours. Nature reviews.    Immunology 12(4):253-268.-   23. Couper K N, et al. (2009) Anti-CD25 antibody-mediated depletion    of effector T cell populations enhances susceptibility of mice to    acute but not chronic Toxoplasma gondii infection. J Immunol    182(7):3985-3994.-   24. Setiady Y Y, Coccia J A, & Park P U (2010) In vivo depletion of    CD4+FOXP3+Treg cells by the PC61 anti-CD25 monoclonal antibody is    mediated by FcgammaRIII+phagocytes. Eur J Immunol 40(3):780-786.-   25. Srivastava M K, et al. (2012) Myeloid suppressor cell depletion    augments antitumor activity in lung cancer. PLoS One 7(7):e40677.-   26. Schmid M C, et al. (2011) Receptor tyrosine kinases and    TLR/IL1Rs unexpectedly activate myeloid cell PI3kgamma, a single    convergent point promoting tumor inflammation and progression.    Cancer Cell 19(6):715-727.-   27. Schmidt-Kittler 0, et al. (PI3Kalpha inhibitors that inhibit    metastasis. Oncotarget 1(5):339-348.-   28. Mandelker D, et al. (2009) A frequent kinase domain mutation    that changes the interaction between PI3Kalpha and the membrane.    Proc Natl Acad Sci USA 106(40):16996-17001.-   29. Zheng Z, et al. (2012) Definition of the binding mode of a new    class of phosphoinositide 3-kinase alpha-selective inhibitors using    in vitro mutagenesis of non-conserved amino acids and kinetic    analysis. Biochem J 444(3):529-535.-   30. Dokmanovic M, Clarke C, & Marks P A (2007) Histone deacetylase    inhibitors: overview and perspectives. Mol Cancer Res 5(10):981-989.-   31. Khan 0 & La Thangue N B (2012) HDAC inhibitors in cancer    biology: emerging mechanisms and clinical applications. Immunology    and cell biology 90(1):85-94.-   32. Lyko F & Brown R (2005) DNA methyltransferase inhibitors and the    development of epigenetic cancer therapies. J Natl Cancer Inst    97(20):1498-1506.-   33. Griffiths E A & Gore S D (2008) DNA methyltransferase and    histone deacetylase inhibitors in the treatment of myelodysplastic    syndromes. Semin Hematol 45(1):23-30.-   34. Baylin S B & Jones P A (2011) A decade of exploring the cancer    epigenome—biological and translational implications. Nat Rev Cancer    11(10):726-734.-   35. Wrangle J, et al. (2013) Alterations of immune response of    non-small cell lung cancer with Azacytidine. Oncotarget    4(11):2067-2079.-   36. Juergens R A, et al. (2011) Combination epigenetic therapy has    efficacy in patients with refractory advanced non-small cell lung    cancer. Cancer Discov 1(7):598-607.-   37. Pulaski B A, Ostrand-Rosenberg S. Mouse 4T1 breast tumor model.    Current protocols in immunology/edited by John E Coligan [et al].    2001; Chapter 20:Unit 20 2.-   38. Youn J I, Collazo M, Shalova I N, Biswas S K, Gabrilovich D I.    Characterization of the nature of granulocytic myeloid-derived    suppressor cells in tumor-bearing mice. Journal of leukocyte    biology. 2012; 91:167-81.-   39. Hamilton M J, Banath J P, Lam V, Lepard N E, Krystal G,    Bennewith K L. Serum inhibits the immunosuppressive function of    myeloid-derived suppressor cells isolated from 4T1 tumor-bearing    mice. Cancer immunology, immunotherapy: CII. 2012; 61:643-54.

1-66. (canceled)
 67. A method of treating a mammal with a bacterial orviral infection, comprising: administering at least one agent whichsuppresses myeloid derived suppressor cells (MDSCs), wherein the agentis selected from the group consisting of a histone deacetylaseinhibitor, a DNA methyltransferase inhibitor, a p110α subunit ofphosphoinositol 3 kinase (PI3K) inhibitor, and combinations thereof. 68.The method of claim 67, wherein the agent is a histone deacetylaseinhibitor and the histone deacetylase inhibitor is entinostat.
 69. Themethod of claim 67, wherein the agent is a DNA methyltransferaseinhibitor and the DNA methyltransferase inhibitor is 5-azacytadine. 70.The method of claim 67, wherein the agent is a p110α subunit ofphosphoinositol 3 kinase (PI3K) inhibitor and the p110α subunit ofphosphoinositol 3 kinase (PI3K) inhibitor is J32.
 71. The method ofclaim 67, wherein the infection is a chronic viral infection.
 72. hemethod of claim 67, wherein the infection is bacterial with associatedsepsis.